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Engineering of Pichia pastoris for improved production of antibody fragments
Biotechnology and bioengineering, 2006-06, Vol.94 (2), p.353-361
Gasser, Brigitte
Maurer, Michael
Gach, Johannes
Kunert, Renate
Mattanovich, Diethard
2006
Volltextzugriff (PDF)
Details
Autor(en) / Beteiligte
Gasser, Brigitte
Maurer, Michael
Gach, Johannes
Kunert, Renate
Mattanovich, Diethard
Titel
Engineering of Pichia pastoris for improved production of antibody fragments
Ist Teil von
Biotechnology and bioengineering, 2006-06, Vol.94 (2), p.353-361
Ort / Verlag
Hoboken: Wiley Subscription Services, Inc., A Wiley Company
Erscheinungsjahr
2006
Quelle
Wiley-Blackwell Journals
Beschreibungen/Notizen
The methylotrophic yeast Pichia pastoris has been used for the expression of many proteins, including antibody fragments. However, limitations became obvious especially when secreting heterodimeric Fab fragments. Up‐to‐date, antibody fragments have only been expressed under control of the strong inducible alcohol oxidase 1 (AOX1) promoter, which may stress the cells by excessive transcription. Here, we examined the secretion characteristics of single chain and Fab fragments of two different monoclonal anti‐HIV1 antibodies (2F5 and 2G12) with both the AOX1 and the glyceraldehyde‐3‐phosphate dehydrogenase (GAP) promoter. Also, the influences of different secretion leaders and strains were evaluated. Interestingly, secretion was only achieved when using the GAP promoter and the Saccharomyces cerevisiae mating factor α (MFα leader), whereas there was no difference between the two P. pastoris strains. During fed batch fermentation of a 2F5 Fab expressing strain, intracellular retention of Fab heavy chains was observed, while both intact Fab and single light chain molecules were only detected in the supernatants. This led to the conclusion that protein folding and heterodimer assembly in the ER are rate limiting steps in Fab secretion. To alleviate this limitation, S. cerevisiae protein disulfide isomerase (PDI) and the unfolded protein response (UPR) transcription factor HAC1 were constitutively overexpressed in P. pastoris. While the overexpression of HAC1 led to a moderate increase of Fab secretion of 1.3‐fold, PDI enabled an increase of the Fab level by 1.9‐fold. Hence, the formation of interchain disulfide bonds can be seen as a major rate limiting factor to Fab assembly and subsequent secretion. © 2006 Wiley Periodicals, Inc.
Sprache
Englisch
Identifikatoren
ISSN: 0006-3592
eISSN: 1097-0290
DOI: 10.1002/bit.20851
Titel-ID: cdi_proquest_miscellaneous_67929323
Format
–
Schlagworte
Alcohol Oxidoreductases - genetics
,
Antibodies, Fungal - biosynthesis
,
Antibodies, Monoclonal - metabolism
,
antibody
,
Basic-Leucine Zipper Transcription Factors - genetics
,
Biological and medical sciences
,
Biotechnology
,
Enzymes
,
Fab
,
Fermentation
,
Fundamental and applied biological sciences. Psychology
,
Fungal Proteins - genetics
,
Gene expression
,
Glyceraldehyde-3-Phosphate Dehydrogenases - genetics
,
Immunoglobulin Fab Fragments - biosynthesis
,
Immunoglobulins
,
Mating Factor
,
PDI
,
Peptides - genetics
,
Pichia - genetics
,
Pichia - metabolism
,
Pichia pastoris
,
Promoter Regions, Genetic
,
Protein Disulfide-Isomerases - genetics
,
Protein Engineering - methods
,
Protein Folding
,
Proteins
,
Repressor Proteins - genetics
,
Saccharomyces cerevisiae
,
Saccharomyces cerevisiae - enzymology
,
Saccharomyces cerevisiae - genetics
,
Saccharomyces cerevisiae Proteins - genetics
,
single chain
,
UPR
,
Yeast
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